Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i>
The green alga <i>Chlamydomonas reinhardtii</i> was recently been shown to be an effective bio-manufacturing platform for the production of recombinant proteins. The advantage of using <i>C. reinhardtii</i> is that it is fast to grow, inexpensive to culture, and relatively sa...
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MDPI AG
2018-12-01
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| Series: | Marine Drugs |
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| Online Access: | https://www.mdpi.com/1660-3397/16/12/493 |
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| author | Jun Chen Jinxia Wu Qingyu Wu Zhangli Hu |
| author_facet | Jun Chen Jinxia Wu Qingyu Wu Zhangli Hu |
| author_sort | Jun Chen |
| collection | DOAJ |
| description | The green alga <i>Chlamydomonas reinhardtii</i> was recently been shown to be an effective bio-manufacturing platform for the production of recombinant proteins. The advantage of using <i>C. reinhardtii</i> is that it is fast to grow, inexpensive to culture, and relatively safe. However, the expression of foreign proteins is always low and difficult to purify in <i>C. reinhardtii</i>. Human kallikrein has the potential to be developed into certain drugs, like insulin. Therefore, its biosynthesis is important to drug development. In this study, we synthesized the <i>sg</i> gene, a signal peptide sequence of alkaline phosphatase, and inserted it into a pH124 plasmid, which contains a <i>HSP70A-RBCS2</i> promoter and a <i>RBCS2</i> terminator. Then, we inserted the human kallikrein gene <i>klk1</i> behind the <i>sg</i> sequence to make a pHsgk124 vector. The pHsgk124 were transferred into a cell-wall deficient strain of <i>C. reinhardtii</i>, cc-503, by using the glass bead method. Southern blot analysis showed that <i>sg</i> and <i>klk1</i> were incorporated into genes of the transgenic <i>C. reinhardtii</i>. RT-PCR analysis showed that it had an active transcription and its expression increased three times under heat stress. Western blot analyses of proteins inside and outside cells (in the culture medium) showed that <i>klk1</i> was expressed in the cell and the resulting protein was secreted into medium. An enzyme activity assay showed that the recombinant protein had the ability to hydrolyze the specific substrate H-D-Val-Leu-Arg-Pna. In conclusion, we successfully bioengineered <i>C. reinhardtii</i> to produce and secrete human kallikrein protein, which has important biomedical implications. |
| first_indexed | 2024-04-11T11:56:54Z |
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| last_indexed | 2024-04-11T11:56:54Z |
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| series | Marine Drugs |
| spelling | doaj.art-d5091a9ef73a42f4b2c01039b1cc72ae2022-12-22T04:25:07ZengMDPI AGMarine Drugs1660-33972018-12-01161249310.3390/md16120493md16120493Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i>Jun Chen0Jinxia Wu1Qingyu Wu2Zhangli Hu3Guangdong Technology Research Center for Marine Algal Bioengineering, Guangdong Key Laboratory of Plant Epigenetic, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaGuangdong Technology Research Center for Marine Algal Bioengineering, Guangdong Key Laboratory of Plant Epigenetic, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaShenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Shenzhen Engineering Laboratory for Marine Algal Biotechnology, College of Life Science and Oceanography, Shenzhen University, Shenzhen 518060, ChinaGuangdong Technology Research Center for Marine Algal Bioengineering, Guangdong Key Laboratory of Plant Epigenetic, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaThe green alga <i>Chlamydomonas reinhardtii</i> was recently been shown to be an effective bio-manufacturing platform for the production of recombinant proteins. The advantage of using <i>C. reinhardtii</i> is that it is fast to grow, inexpensive to culture, and relatively safe. However, the expression of foreign proteins is always low and difficult to purify in <i>C. reinhardtii</i>. Human kallikrein has the potential to be developed into certain drugs, like insulin. Therefore, its biosynthesis is important to drug development. In this study, we synthesized the <i>sg</i> gene, a signal peptide sequence of alkaline phosphatase, and inserted it into a pH124 plasmid, which contains a <i>HSP70A-RBCS2</i> promoter and a <i>RBCS2</i> terminator. Then, we inserted the human kallikrein gene <i>klk1</i> behind the <i>sg</i> sequence to make a pHsgk124 vector. The pHsgk124 were transferred into a cell-wall deficient strain of <i>C. reinhardtii</i>, cc-503, by using the glass bead method. Southern blot analysis showed that <i>sg</i> and <i>klk1</i> were incorporated into genes of the transgenic <i>C. reinhardtii</i>. RT-PCR analysis showed that it had an active transcription and its expression increased three times under heat stress. Western blot analyses of proteins inside and outside cells (in the culture medium) showed that <i>klk1</i> was expressed in the cell and the resulting protein was secreted into medium. An enzyme activity assay showed that the recombinant protein had the ability to hydrolyze the specific substrate H-D-Val-Leu-Arg-Pna. In conclusion, we successfully bioengineered <i>C. reinhardtii</i> to produce and secrete human kallikrein protein, which has important biomedical implications.https://www.mdpi.com/1660-3397/16/12/493<i>Chlamydomonas reinhardtii</i>human kallikreinsignal peptidesecretory vector |
| spellingShingle | Jun Chen Jinxia Wu Qingyu Wu Zhangli Hu Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i> Marine Drugs <i>Chlamydomonas reinhardtii</i> human kallikrein signal peptide secretory vector |
| title | Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i> |
| title_full | Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i> |
| title_fullStr | Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i> |
| title_full_unstemmed | Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i> |
| title_short | Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic <i>Chlamydomonas reinhardtii</i> |
| title_sort | biosynthesis and secretion of human tissue kallikrein in transgenic i chlamydomonas reinhardtii i |
| topic | <i>Chlamydomonas reinhardtii</i> human kallikrein signal peptide secretory vector |
| url | https://www.mdpi.com/1660-3397/16/12/493 |
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